Note: Descriptions are shown in the official language in which they were submitted.
~8~i07~
4-85
RNA TRANSFORMATION VECTOR
Field of the Invention
This invention relates to the field of plant viruses, more particularly
to (+) s~rand RNA viruses of plants,animals and bacteria, and to modifica-
tions, made according to the teachings herein, which permit insertion of an
exogenous RNA segment into the viral genome, The inserted segment can then be
introduced into a host cell in order to modify the cell, either genotypically
or phenotypically. The invention is exemplified by modifications of an RNA
plant virus, brome mosaic virus (8MV), which is infective for monocots.
Background _ Prior Ar
RNA viruses whose genome is composed of a single RNA strand capable of
replication in the cytoplasm of a host by direct RNA replication are wide-
spread, many varieties of which are known and which infect animals, plants and
bacteria. Such viruses are sometimes termed "(+) strand RNA viruses" since
the infective RNA strand, that normally found encapsidated in the virus parti-
cle, is a messenger-sense strand, capable of being directly translated, and
also capable of being replicated under the proper conditions by a direct
process of RNA replication. Viruses belonging to this group include, but are
not limited to, the picornaviruses, the RNA bacteriophages, the comoviruses,
and various single component and multicomponent RNA viruses of plants. A
partial listing of such viruses would include polio virus, sindbis virus, Q~
bacteriophage, tobacco mosaic virus, barley stripe mosaic virus, cow pea
mosaic virus, cucumber mosaic virus, alfalfa mosaic virus and brome mosaic
virus. In some cases, the entire virus genome is contained within a single
RNA molecule, while in other cases, most notably the multicomponent RNA plant
viruses, the total genome of the virus consists of two or more distinct RNA
segments, each separately encapsidated. (For general review, see General
Virology, S. Luria and J. Darnell; _ ant Yirology 2nd ed., R. E. F. Matthews,
Academic Press (1981); and for a general review of (+) strand RNA replication,
see Davies and Hull (1982) J. Gen. Virol. 61,1). Within the group there are
~8~
wide variations in capsid morphology, coat proteins, genetic organization and
genome size.
Despite the well-documented diversity, recent studies have shown striking
similarities between the proteins which function in RNA replication. Sequence
homo~ogies have been reported between the cowpea mosaic virus, poliovirus and
foot-and-mouth disease virus (Franssen, H. (1984) EM30 Journal 3,855), between
non-structural proteins encoded by alfalfa mosaic virus, brome mosaic virus
and tobacco mosaic virus, Haseloff, J. et al. (1984), Proc. Nat. Acad. Sci.
USA 81, 4358, and between the same proteins and proteins encoded by sindbis
virus, Ahlquist, P. et al. (1985) J. Virol. 5~, 536. Evidence of such sub-
stantial homology in proteins related to the replication functions indicate
that the viruses share mechanistic similarities in their replication strate-
gies and may actually be evolutionarily related. In the present inven~ion,
modifications to the genomic RNA of a (~) strand RNA virus are disclosed. The
modified RNA is usëd to transfer a desired RNA segment into a targeted host
cell and to replicate that segment and express its function within the host
cell. A virus known to be representative of the common replication functions
of (+) strand RNA viruses was chosen to exemplify the present invention here-
n.
Brome mosaic virus (BMV) is one member of a class of plant viruses char-
acterized by a multipartite RNA genome. The genetic material of the virus is
RNA, and the total genetic information reqwired for replication and productive
infection is divided into more than one discrete RNA molecule. The class,
termed multipartite RNA viruses herein, includes, besides BMV, such viruses as
alfalfa mosaic virus (AMV), barley stripe mosaic virus, cowpea mosaic virus,
cucumber mosaic virus, and many others. Virus particles are generally com-
posed of RNA encapsidated by a protein coat. The separate RNA molecules which
comprise the total genome of a given multipartite virus are encapsidated in
separate virus particles, each of which has the same protein composition.
Infection of a host plant cell occurs when a virus particle containing each of
the RNA components of the viral genome has infected the cell, for example by
exposing a plant to a virus preparation containing a mixture of all necessary
viral components. Infection may also be achieved by exposing a plant cell or
protoplast to a mixture of the RNA components. A subclass of the multipartite
RNA viruses ttermed subclass I herein) requires coat protein in addition to
viral RNA for replication and productive infection. AMV is an example of a
. - ..,,,... ~ - .
,.
~ 2~3073
subclass I multipartite virus. Another subclass (termed subclass II herein)
does not require coat protein, the component RNAs being both necessary and
sufficient for replication and productive infection. BMV belongs to subclass
II. The BMV genome is divided among three messenger-sense RNAs of 3.2, 2.8
and 2.1 kilobases (Ahlquist, P. et al. (1981) J. Mol. Biol. 153,23; Ahlquist,
P., et al. (1984) J. Mol. Biol. 172,369). The term "messenger-sense" denotes
that the viral RNAs can be directly translated to yield viral proteins, with-
out the need for an intervening transcription step.
Complete cDNA copies of each of the three BMV genetic components have
been cloned in a general transcription vector, pPM1, described by Ahlquist, P.
and Janda, M. (1984) Mol. Cell Biol. 4,2976. Three plasmids have been se-
lected, pBlPM18, pB2PM25 and pB3PM1 containing, respectively, cDNA copies of
BMY-RNA1, BMV-RNA2 and BMV-RNA3. The three plasmids constitute, as a set, the
complete BMV genome.
DNA from each of the three BMY cDNA-containing plasmids can be cleaved at
a unique EcoRI site. The linear DNA thus produced can be transcribed in vitro
in a reaction catalyzed by RNA polymerase. A modified ~ PR promoter in the
transcription vector, pPM1, allows RNA synthesis to initiate exactly at the 5'
terminus of each BMV sequence, and transcription continues to the end of the
DNA template, adding 6-7 nonviral nucleotides at the 3' ends of the tran-
scripts. When transcription is carried out in the presence of a synthetic cap
structure, m7GpppG, as described by Contreras, R., et al. (1982) Nucleic Acids
Res. 10,6353, RNA transcripts are produced with the same capped 5' ends as
authentic BMV RNAs. These RNAs are active messengers in in vitro translation
systems and direct production of proteins with the same electrophoretic mobil-
ities as those translated from authentic BMV RNAs.
Summary of the Invention - For the sake of brevity, the term "RNA virus" is
used herein to mean (+) strand replicating RNA viruses.
The invention is based on the discovery that an RNA of the genome of an
RNA virus can be modified to include an exogenous RNA segment and that the
modified RNA can be introduced into a host cell, replicated therein and can
express the exogenous RNA segment. The recipient cell is thereby phenotypi-
cally transfonmed and may contribute to a genotypically transformed organism,
as well. Phenotypically transformed cells can be modified in vivo, in planta,
~2~S0'73
in tissue culture, in cell culture or in the form of protoplasts. The exem-
plified embodiment of the invention is useful for producing phenotypically
transformed plants under field conditions or greenhouse growth. Traits desir-
able for introduction in this manner include, but are not limited to, pest
resistance, pathogen resistance, herbicide tolerance or resistance, modified
growth habit and modified metabolic characteristics, such as the production o~
commercially useful peptides or pharmaceuticals in plants. The modifications
can be applied at any time during the growth cycle, depending on the need for
the trait. For example, resistance to a pest could be conferred only if the
crop were at risk for that pest, and at the time when the crop was most likely
to be affected by the pest. Other traits can be used to enhance secondary
properties, for example to increase the protein content of post-harvest for-
age. Any plant variety susceptible to infection by a multipartite RNA virus
can be phenotypically transformed. The choice of virus and the details of
modification will be matters of choice depending on parameters known and
understood by those of ordinary skill in the art. Other uses for cells and
organisms phenotypically or genotypically modified by means of a modified RNA
derived from an RNA virus will be readily apparent to those skilled in the
art, given a wide range of RNA viruses to modify and a wide range of suscepti-
ble host cell types. Other uses for transformed animal cells, bacterial cells
and the like can be readily envisioned. For example, bacterial cells suscep-
tible to Q~ phage can be grown in culture to desired cell density, infected
with a modified Q~ phage carrying a desired gene and thereby caused to express
large quantities of a desired protein within a short time period.
Generally, the steps of a process for phenotypically transforming a cell
or organism are: forming a full-length cDNA transcript of the virus RNA, or
of each RNA component if the RNA virus is multipartite; cloning each cDNA in a
transcription vector; modifying the cDNA of at least one of the RNA components
by inserting a non-viral DNA segment in a region able to tolerate such inser-
tion without disrupting RNA replication thereof; transcribing the modified
cDNA, or, in the case of a multipartite virus, transcribing each cDNA corre-
sponding to an RNA component of the multipartite virus; substituting the
modified cDNA for its unmodified counterpart in the transcription reaction;
infecting virus-susceptible protoplasts, cells, tissues or whole organisms
with transcribed RNA, or a mixture of RNAs, either in solution or encapsi-
dated, o~ each viral component including the modified RNA comprising messen-
ger-sense RNA containing an exogenous RNA segment. From this point, the steps
38073
to be followed will vary, depending on the type of
material infected and the route of infection.
Protoplasts, cells and tissues of plants can be
propagated vegetatively, regenerated to yield whole
plants by means of any technique suitable to the
particular plant variety infected, and transplanted to
the field. Whole plants can be infected in situ.
Infected plants and plant cells can produce many copies
per cell of the modified viral RNA containing the
exogenous RNA segment. If desired and if suitably
inserted, by means of principles and processes known in
the art, the exogenous RNA segment can be caused to
carry out a function within the cell. Such a function
could be a coding function, translated within the cell
to yield a desired peptide or protein, or it could be a
regulatory function, increasing, decreasing, turning on
or off the expression of certain genes within the cell.
Any function which a segment of RNA is capable of
providing can, in principle, be expressed within the
cell. The exogenous RNA segment thus expressed confers
a new phenotypic trait to the transformed organism,
plant, cells, protoplasts or tissues.
The invention is exemplified herein by the
modification of BMV RNA to contain a structural gene
encoding chloramphenicol acetyl transferase (CAT) and
the phenotypic modification of barley protoplasts
therewith, yielding protoplasts synthesizing CAT. The
data presented herein are believed to represent the
first instance of phenotypic modification of a cell by
means of a modified RNA of an RNA virus.
In one aspect of present invention, there is
provided a capped RNA molecule capable of infecting a
host cell, which capped molecule comprises a cls-acting
replication element derived from a (+) strand RNA virus,
which capped molecule has no extraneous nonviral
sequences between the cap site and the 5' terminus of
., ~i
~ ~288~73
5A
the viral sequence, and which capped molecule further
comprises an exogenous RNA segment capable of expressing
its function in a host cell in a region of the capped
RNA molecule able to tolerate the segment without
disrupting RNA replication of the capped RNA molecule,
in the presence of trans-acting replication elements in
said host cell.
Detailed Description of the Invention
In order to facilitate understanding of the
invention, certain terms used throughout are herein
defined.
RNA virus - The term as used herein means a virus
whose genome is RNA in single-stranded form, the
single strand being a (+) strand, or
messenger-sense strand. Replication of the viral
(+) strand in a virus-infected cell occurs by a
process of direct RNA replication and is therefore
distinguishable from the replication mechanism of
retroviruses which undergo an intermediate step of
reverse transcription in the host cell.
~; .
. . ,
1~8807~
Cis-acting replication element - This term denotes that
portion of the RNA genome of an RNA virus which must be
present in cis, that is, present as part of each viral
strand as a necessary condition for replication. Virus
replication presumably depends upon the existence of one
or more trans (diffusible) elements which interact with
the Ci s-acting element to carry out RNA replication.
While trans-acting elements are necessary for replica-
tion, they need not be present or coded for on the modi-
fied RNA provided they are made available within the
infected cell by some other means. For example, in the
case of a mulitpartite RNA virus, the trans-acting func-
tions may be provided by other, unmodified components of
the viral genome used to transform the cells simultane-
ously with the modified RNA. The target cell may also be
modified in a previous step to provide constitutive
expression of the trans-acting functions. The cis-acting
replication element is composed of one or more segments
of viral RNA which must be present on any RNA molecule
that is to be replicated within a host cell by RNA repli-
cation. The segment will most likely be the 5' terminal
portion of the viral RNA molecule, and may include other
portions as well. The cis-acting element is therefore
defined in functional terms: any modification which
destroys the ability of the RNA to replicate in a cell
known to contain the requisite trans-acting elements, is
deemed to be a modification in the cis-acting replication
element. Conversely, any modification, such as an inser-
tion in a sequence region which is able to tolerate such
insertion without disrupting replication, is a modifica-
tion outside the cis-acting replication element. As is
demonstrated herein, using the example of BMV, substan-
tial portions of an RNA virus molecule may be modified,
by deletion, insertion, or by a combination of deletion
and insertion, without disrupting replication.
The term "derived from" is used to identify the viral
1281~0~3
source of an RNA segment which comprises part of the
modified RNA. For example, for the modified RNAs
described herein, substantial portions thereof are
derived from BMV. The manner of deriving, whether by
direct recombination at the RNA level, by transcription
or by reverse transcription does not matter for the
purpose of the invention. Indeed, it is contemplated
that modifications may be made within the cis-acting
replication element and elsewhere for example to modify
the rate or amount of replication that is obtained. In
the case of modified RNAs exemplified herein, a tran-
scription vector was employed which preserved the exact
5' terminal nucleotide sequence of viral RNA. However
the use of such a vector in transcribing viral RNA from
cDNA is not considered essential to the invention,
although it will be preferred if preservation of the
exact nucleotide sequence at the 5' end is desired. An
RNA segment which has been derived from a given source
virus may, but need not be, identical in sequence to that
segment as it exists in the virus. It will be understood
that a cis-acting replicating element derived from a
given RNA virus may have minor modifications in the
nucleotide sequence thereof without substantially inter-
fering with RNA replication.
Exogenous RNA segment is a term used to describe a seg-
ment of RNA to be inserted into the virus RNA to be
modified, the source of the exogenous RNA segment being
different from the RNA virus itself. The source may be
another virus, a living organism such as a plant, animal,
bacteria, virus or fungus, the exogenous RNA may be a
chemically synthesized RNA or it may be a combination of
the foregoing. The exogenous RNA segment may provide any
function which is appropriate and known to be provided by
an RNA segment. Such functions include, but are not
limited to, a coding function in which the RNA acts as a
messenger RNA encoding a sequence which, translated by
` ~l288{~3
the host cell, results in synthesis of a peptide or
protein having useful or desired properties; the RNA
segment may also be structural, as for example in riboso-
mal RNA, it may be regulatory, as for example with small
nuclear RNAs or anti-sense RNA, or it may be catalytic.
A particularly interesting function is provided by anti-
sense RNA, sometimes termed (-) strand RNA, which is in
fact a sequence complementary to another RNA sequence
present in the target cell which can, through complemen-
tary base pairing, bind to and inhibit the function of
the RNA in the target cell,
Various aspects of the stages outlined in the Summary section can be
modified as needed, depending upon specific aspects of the virus selected as
the transforming agent and of the RNA segment to be inserted. For example, if
the inserted gene is in the form of messenger-sense RNA to be directly trans-
lated by the transformed cell, the gene must be free of intervening, non-
translated sequences, such as introns. On the other hand, the inserted gene
need not be a naturally occurring gene, but may be modified, a composite of
more than one coding segment, or it may encode more than one protein. The RNA
may also be modified by combining insertions and deletions in order to control
the total length or other properties of the modified RNA molecule. As demon-
strated in Example 5, a substantial portion of the RNA3 of BMY can be deleted
without significantly effecting its replication in cells containing normal
RNA1 and RNA2. The inserted non-viral gene may be either prokaryotic or
eukaryotic in origin as long as it is in a form which can be directly trans-
lated by the translation machinery of the recipient cell. Eukaryotic genes
containing introns within the coding sequence must therefore be inserted in
the form of a cDNA copy of the eukaryotic messenger RNA encoding the gene.
The inserted gene may contain its own translation start signals, for example,
a ribosomal binding site and start (AUG) codon, or it may be inserted in a
manner which takes advantage of one or more of these components preexisting in
the viral RNA to be modified. Certain structural constraints must be observed
to preserve correct translation of the inserted sequence, according to princi-
ples well understood in the art. For example, if it is intended that the
exogenous coding segment is to be combined with an endogenous coding segment,
the coding sequence to be inserted must be inserted in reading frame phase
~l288073
therewith and in the same translational direction. The tenm "non-viral`' is
used herein in a special sense to include any RNA segment which is not nor-
mally contained within the virus whose modification is exploited for effecting
gene transfer and is therefore used synonymously with "exogenous". Therefore,
a gene derived from a different virus species than that modified is included
within the meaning of the terms "non-viral" and "exogenous" for the purposes
of describing the invention. For example, a non-viral gene as the term is
used herein could include a gene derived from a bacterial virus, an animal
virus, or a plant virus of a type distinguishable from the virus modified to
effect transformation. In addition, a non-viral gene may be a structural gene
derived from any prokaryotic or eukaryotic organism. It will be understood by
those ordinarily skilled in the art that there may exist certain genes whose
transfer does not result in obvious phenotypic modification of the recipient
cell. Such may occur, for example, if the translation product of the non-
viral gene is toxic to the host cell, is degraded or processed in a matter
which renders it non-functional or possesses structural features which render
it impossible for the host cell to translate in sufficient quantities to
confer a detectable phenotype on the transformed cells. However, the inven-
tion does not depend upon any specific property of an RNA segment or gene
being transferred. Therefore, the possible existence of RNA segments or genes
which fail to confer a readily observalbe phenotypic trait on recipient cells
or plants is irrelevant to the invention and in any case will be readily
recognizable by those of ordinary skill in the art without undue experimenta-
tion.
An exogenous RNA segment may be inserted at any convenient insertion site
in any of the cDNA sequences corresponding to a viral RNA, or component RNA of
a multipartite RNA virus, provided the insertion does not disrupt a sequence
essential for replication of the RNA within the host cell. For example, for a
virus whose coat protein is not essential for replication, an exogenous RNA
segment may be inserted within or substituted for the region which normally
codes for coat protein. As desired, regions which contribute to undesirable
host cell responses may be deleted or inactivated, provided such changes do
not adversely effect the ability of the RNA to be replicated in the host
cell. For many single component and multipartite RNA viruses, a reduction in
the rate of normal RNA replication is tolerable and will in some instances be
preferred, since the amount of RNA producéd in a normal infection is more than
enough to saturate the ribosomes of the transformed cell.
88073
The transformation process itself can be carried out by any means whereby
RNA can be introduced into cells, whole plants, plant tissues or proto-
plasts. Host cells can be infected by the RNA alone, or encapsidated in a
virus particle, except that the modified viral RNA containing a non-viral RNA
segment is substituted for its counterpart in a normal infection. Any other
suitable means for introducing RNA into target cells such as microinjection
may be used. In some cases it may be preferable to include all of the normal
components in addition to the modified component. More than one component may
be modified in the mixture of transforming components. ~t will be understood
that the amounts of each infecting component must be sufficient to insure that
an adequate number of cells receive at least one of each component in the
mixture. Other variables of the infection process, such as pretreatment of
the recipients, addition of components to enhance the efficiency of infection,
use of encapsidated or unencapsidated RNA, are matters of choice which those
of ordinary skill in the art will be able to manipulate to achieve desired
transformation efficiency in a given situation. For instance, the choice of
multipartite plant RNA virus to be modified to effect gene transfer in a given
plant variety will depend upon known host range properties of multipartite RNA
viruses. For example, BMV infects a variety of grasses and their related
domesticated relatives including barley, wheat and maize.
Plant cells which are infected in culture will normally remain trans-
formed as the cells grow and divide since the RNA components are able to
replicate and thus become distributed to daughter cells upon cell division.
Plants regenerated from phenotypically modified cells, tissues or protoplasts
remain phenotypically modified. Similarly, plants transformed as seedlings
remain transfonmed during growth. Timing of application of the transforming
components will be governed by the result which is intended and by variations
in susceptibility to the transforming components during various stages of
plant growth.
Many plant RNA viruses are seed transmitted from one generation to the
next. This property can be exploited to effect genotypic transformation of a
plant. That is to say~ the modified RNA remains transmissible from one gener-
ation to the next, most likely by replication in the cytoplasm, and thereby
becomes transmissible from one generation to the next, just as seed-borne
virus infections are transmitted from one generation to the next.
~:8~073
11
The following examples illustrate the principles of the invention as
applied to modification of BMV RNA3 and the use of modified BMV RNA3 contain-
ing a gene coding for chloramphentcol acetyl transferase (CAT) in the pheno-
typic transformation of barley protoplasts. For convenience, any modification
to a viral RNA which includes the insertion of a nonviral ribonucleotide
sequence, whether or not combined with a deletion of viral RNA will be desig-
nated by a prime symbol following the number designating the RNA. For
example, modified RNA3 is termed RNA3', or more generally, RNAn is designated
RNAn'.
The following examples utilize many techniques well known and accessible
to those skilled in the arts of molecular biology, cloning, plant cell
biology, plant virology and plant tissue culture. Such methods are fully
described in one or more of the cited references if not described in detail
herein. Unless specified otherwise, enzymes were obtained from commercial
sources and were used according to the vendor's recommendations or other
variations known to the art. Reagents, buffers and culture conditions and
reaction conditions for various enzyme catalyzed reactions are also known to
those in the art. Reference works containing such standard techniques include
the following: R. Wu, ed. (1979) Meth. Enzymol. 68; R. Wu et al., eds. (1983)
Meth. Enzymol. 100, 101; L. ~rossman and K. Moldave, eds. (1980) Meth.
Enzymol. 65; J. H. Miller (1972) ExPeriments in Molecular Genetics; R. Davis
et al. (1980) Advanced Bacterial Genetics; R. F. Schleif and P. C. Wensink
(1982) Practical Methods ~n Molecular Biology; and T. Manniatis et al. (1982)
Molecular Cloning.
Textual use of the name of a restriction endonuclease in isolation, e.g.,
"BclI" refers to use of that enzyme in an enzymatic digestion, except in a
diagram where it can refer to the site of a sequence susceptible to action of
that enzyme, e.g., a restriction site. In the text, restriction sites are
indicated by the additional use of the word "site", e.g., "BclI site". The
additional use of the word "fragment", e.g., "BclI fragment", indicates a
linear double-stranded DNA molecule having ends generated by action of the
named enzyme (e.g., a restriction fragment). A phrase such as "BclItSmaI
fragment" indicates that the restriction fragment was generated by the action
of two different enzymes, here BclI and SmaI, the two ends resulting from the
action of different enzymes. Note that the ends will have the characteristics
of being either sticky (i.e.~ having a single strand of protrusion capable of
1~813073
-
l?
base-pairing with a complementary single-stranded oligonucleotide) or blunt
(i.e., having no single-stranded protrusion) and that the specificity of a
sticky end will be detenmined by the sequence of nucleotides comprising the
single-stranded protrusion which in turn is determined by the specificity of
the enzyme which produces it.
All plasmids are designated by a sequence of letters and numbers prefaced
by a lower case "p", for example, pPMl. Clones of complete BMV cDNA inserted
in pPM1 are named by the format pBxPMy, where x equals 1, 2 or 3 designating
the BMV component cloned (i.e., from RNAl, 2 or 3) and y is an arbitrary
isolate number. Thus, the set of three plasmids, pBlPM18, pB2PM25 and pB3PMl
contains complete cDNA copies of BMV RNAs 1~ 2 and 3, respectively, and repre-
sent, as a set, the complete BMV genome. Certain steps of cloning, selection
and vector increase employed strains of E. coli. While the strains used
herein have been designated, there are many equivalent strains available to
the public which may be employed. The use of a particular microorganism as a
substitute for a strain designated herein is a matter of routine choice avail-
able to those of ordinary skill in the art, according to well-known princi-
ples.
13
Example 1: Infectivity of transcribed BMV-cDNA
In vitro Transcription. Transcription reactions contained 25mM Tris-HCl,
pH 8.0/5 mM MgC12/150 mM NaCl/1 ~M dithiothreitol/200 ~M each rATP, rCTP, and
rUTP/25~M rGTP/500~M m7GpppG (P-L Biochemicals)/plasmid DNA (0.1 ~g/~l)
Escherichia coli RNA polymerase (0.05 units/~l) (Promega Biotec, Madison,
WI). Reactions were incubated 30 minutes at 37C, by which time the rGTP was
nearly exhausted. Additional rGTP was added to 25~M and incubation continued
a further 30 minutes. For uncapped transcripts, m7GpppG was deleted, rGTP was
increased to 200~M, the concentrations of DNA and polymerase were doubled, and
incubation was carried out for 1 hour. Reactions were stopped by addition of
EDTA to 10mM and either diluted directly in inoculation buffer or phenol-
extracted before nucleic acid recovery by ethanol precipitation. In most
experiments, plasmids representing all three BMV components were pooled and
cleaved at unique EcoRI sites 3 base pairs past the 3' terminus of each BMV
sequence before transcription. Fig. 1 shows a map of EcoRI-cleaved pB3M1.
The maps for pPM1 containing cDNA of RNA1 or RNA2 are the same, except that
the region labeled "BMV-cDNA" is cDNA of RNA-1 or RNA-2.
Infectivity Testing. Seven-day-old barley seedlings (Hordeum vulgare L.
cv. Morex) were dusted with carborundum powder and inoculated with either
virion RNA or in vitro transcription mixes in 50mM Tris P04, pH 8.0/250 mM
NaCl/5mM EDTA/Bentonite (5 mg/ml) (5); 15-30 plants in a single 14-cm-diameter
pot were treated with the same inoculum, using 10-30 ~l per plant. Plants
were scored for the presence of mosaic symptoms 7-14 days after inoculation.
BMN Isolation. Fourteen days after inoculation, virus was isolated from
barley plants as described by Shih, et al. (1972) J. Mol. Biol. 64,353, with
the substitution of chloroform for carbon tetrachloride and a second polyethy-
lene glycol precipitation for differential centrifugation. Viral RNA was
isolated by phenol extraction and ethanol precipitation.
Infectivity Testing of BMV cDNA Clones and Their in ~Atro Transcripts.
Cloning of complete cDNA copies of all three BMV genetic components in a
general transcription vector, pPM1, has been described by Ahlquist, P. and
Janda, M. (1984) Mol. Cell. Biol. 4,2876. DNA from such clones can be cleaved
with EcoRI (Fig. 1) and transcribed in vitro in the presence of a synthetic
cap structure to produce complete RNA copies of the BMV components that have
the same capped 5' ends as authentic BMV RNAs, and an additional 6-7 non-viral
nucleotides at their 3' ends.
1281~3073
14
To test the infectivity of these cloned DNAs and their transcripts, three
plasmids, pBlPM18, pB2PM25, and pB3PM1, were selected. The selected clones
contain cDNA copies of BMV RNAs 1, 2, and 3~ respectively, and represent, as a
set, the complete BMV genome. The natural isolate of BMV propagated in our
laboratory is referred to by its usual designation of Russian strain. Mix-
tures of the EcoRI-cut M1 plasmids and their capped transcription products
were inoculated onto barley plants in parallel with untranscribed DNA from the
same plasmids. As Judged by the production of normal viral symptoms, the
transcribed plasmid mixture was infectious, while the untranscribed plasmid
mixture was not (Table 1).
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Table 1. Comparison of infectivity of EcoRI-cut M1 plasmids, transcribed
EcoRI-cut M1 plasmids, and Russian strain BMV virion RNAs over a range of
inoculum concentrations.
.
Plants with
Pot No. Inoculum, ng/~l symptoms/total
EcoRI-cut pBlPM18,
pB2PM25,pB3PM1
1 100 0/21
2 10 0/23
3 1 0/22
Transcribed EcoRI-cut
pBlPM18, pB2PM25,
pB3PM1
4 40 19~23
4 7/20
6 0.4 0/21
Russian strain BMV
virion RNA
7 10 21/22
8 1 14/21
9 0.1 2/21
Mock-inoculated
0 0/22
In vitro transcription yields approximately 3 BMV transcripts per plasmid
~Ahlquist and Janda, 1984). Total BMV transcript content of the inocula for
pots 4-6 is thus approximately 75, 7.5, and 0.75 ng/~l, respectively.
~ ~8S~)73
16
The effects of various alterations to the transcription protocol were
examined to more clearly characterize the infectious entity observed in plas-
mid transcription mixes. Infectivity required transcription of clones repre-
senting all three BMV genetic components. Moreover, infectivity was sensitive
to HinfI before or to RNase A after transcription, but it was not signifi-
cantly affected by RNase A before or HinfI after transcription. HinfI cleaves
at 8 sites within pPM1 and at 15, 10, and 12 sites within BMY 1, 2, and 3
cDNAs, respectively. These results confirm that the observed infectivity
arises from the in vitro transcripts rather than directly from their DNA
templates. In addition, when plasmids were either not cut or were cut with
PstI before transcription (cleaving 2.7 kilobases rather than 7 bases down-
stream of the cDNA end), infection was not observed, suggesting that infecti-
vity is affected by the structure of the transcript 3' end. Finally, if the
cap analog was omitted during in vitro transcription, no infection was
detected, even if inoculum concentration was increased 20-fold.
Infectivity of RNA transcribed in vitro from EcoRI-cut M1 plasmids was
clearly lower than that of authentic BMV RNA. The number of infected plants
produced from a given weight of in vitro-transcribed RNA was similar to that
produced from 1/lOth that weight of authentic BMV RNA (Table 1). The presence
of the plasmid DNA template in the inoculum was not responsible for this
effect, as addition of the same plasmid DNA to authentic BMV RNA did not
affect its infectivity.
Correlation of Sympkomology with BMN Replication. To establish that such
symptoms accurately reflect BMV replication, several molecular tests were
applied. Nitrocellulose dot blots of total RNA (described by Garger, S. J. et
al. (1983) Plant Mol. Biol. Reporter 1,21) extracted from leaves of symptom-
expressing and symptomless plants inoculated with either authentic BMV RNA or
in vitro BMV transcripts were probed with 32P-labeled cloned BMV cDNA. In all
cases, symptom-expressing leaves showed a positive hybridization response, and
in all cases but one, symptomless leaves gave a negative response. The one
exception was from a plant that had been inoculated with in vitro transcripts
and showed no visible symptoms but gave a positive hybridization signal.
Virus isolated from plants infected with cDNA transcripts is serologi-
cally identical to Russian strain BMV in double-diffusion tests with anti-BMV
antisera. Phenol extraction of BMV isolated from transcript-infected plants
8S~)73
17
releases four RNAs that comigrate with Russian strain virion RNAs, hybridi2e
to B~lV-specific DNA probes, and are highly infectious in subsequent inocula-
tions. Therefore, multipartite RNA plant virus infection can be derived
solely from appropriately cloned viral cDNA by means of a simple transcription
step.
xample _ Construction and replication of a specific deletion in the BMV_ _ _ _
c gene
In the following example, reference may be made to Fig. 1 for location of
the relevant restriction sites.
Plasmid pB3PM1 DNA (Ahlquist, P. and Janda, M. (1984)) was cleaved with
SalI and XbaI and treated with the Klenow fragment of DNA polymerase I to
generate blunt ends (Maniatis et al. (1982) Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor). The approximately 5.2 kb fragment was isolated
from a low melting point (LMP) agarose gel (Sanger et al. (1980) J. Mol. Biol.
143,161), recircularized by treatment with T4 DNA ligase, and transformed into
competent E. coli JM101. RF DNA from selected ampicillin resistant transfor-
mants was digested simultaneously with SalI and EcoRI to confirm regeneration
of the _ I site and deletion of the desired fragment. A single tested clone,
designated pB3DCP10, having the region of the coat gene from SalI to SbaI
deleted, was selected for further work.
EcoRI-digested pB3DCP10 was transcribed under capping conditions
(Ahlquist and Janda, 1984) along with EcoRI-digested pBlPM18 and pB2PM25
(Ahlquist and Janda, 1984), and the transcripts were separated from the plas-
mid DNA templates by LiCl precipitation (Baltimore (1966) J. Mol. Biol. 18,
421). Barley protoplasts were prepared as described by Loesch-Fries and Hall
~1980) J. Gen. Virol. 47, 323, and inoculated as described by Samac et al.
(1983) Virology 131, 455, with the transcripts and incubated in the presence
of ~3H]uridine. Total nucleic acids were extracted and analyzed on acryla-
mide-agarose gels as described by Loesch-Fries and Hall, 1980. The deleted
RNA3 derived from pB3DCP10 was found to both replicate and generate a deleted
version of subgenomic RNM . (RWM is a subgenomic fragment of RNA3 produced
during infection). This example demonstrates that a substantial portion of
RNA3 encoding coat protein can be deleted, without preventing replication of
viral RNAs.
~ ~88073
ExamPle 3: Insertion of a PstI site at the 3' cDNA end of plasmid pB3PM1
Construction and use of transcribable BMV cDNA clones has been described
before (Ahlquist et al., 1984a, 1984b). To define the transcript 3' end, the
originally described plasmids are first linearized before transcription by
clea~age of an EcoRI site just outside the 3' end of BMV cDNA. However, such
EcoRI cleavage results in addition of 6-7 nonviral nucleotides to the tran-
script and is inconvenient for transcription of BMV-linked foreign sequences
which contain EcoRI site(s). To deal with both of these problems, a PstI
site, i.e., a nucleotide sequence including a sequence recognized and cleaved
by PstI endonuclease, WdS inserted immediately adjacent to the BMV cDNA in
pB3PM2 to provide an alternate cleavage site. The steps in the construction
can be followed by referring to Fig. 2. DNA sequences shown in Fig. 2 are
plus (+) strands only, defined as equivalent (not complementary) to the RNA
sequence of BMV-RNA.
Insertion of this PstI site was generally similar to the previously
described insertion of a SmaI site adjacent to the lambda PR promoter
(Ahlquist and Janda, 1984). First the 0.9 kb SalI-EcoRI fragment of pB3PM1
(Fig. 1) was isolated from a low-melting point agarose gel and subcloned into
SalI EcoRI cleaved M13mp9. Colorless recombinant plaques were selected on X-
gal/IPTG plates and the insertion of BMV sequences verified by dideoxynucleo-
tide sequencing (Biggen et al. (1983) Proc. Nat. Acad. Sci. USA 80, 3963). A
single clone, designated M13/B3ES1, was selected for further work. A 21
nucleotide mismatch primer (Fig. 2) was chemically synthesized and purified
and used to prime synthesis of 32P-labeled DNA from M13/B3ES1 ssDNA. After
synthesis, the DNA was cleaved with AvaI at a site in the M13 vector distal to
the primer and the major labelled DNA fragment, containing the mismatch primer
at its 5' end and BMV3 sequences interior, was purified on an alkaline agarose
low melting point gel (Maniatis et al., 1982). A second strand of DNA was
primed with a lac reverse primer (Ahlquist and Janda, 1984), the ds synthetic
DNA cleaved with XbaI and the approximately 0.36 kb dsDNA fragment, containing
the mismatch primer linked to 3' BMV RNA3 sequences, isolated from a low
melting point agarose gel. This fragment was then subcloned into XbaI-SmaI
cut M13mpl9. Colorless recombinant plaques were selected on X-gal/IPTG plates
and the correct linkage of the PstI site to BMV cDNA confirmed by dideoxy
sequenclng. The 0.36 kb XbaI EcoRI fragment from a selected M13 clone was
~ 2~8073
19
recloned between the XbaI and SalI sites of Pb3PM1, creating plasmid pB3~P1.
The sequence of RNA transcribed from PstI-cleaved pB3nP1 will be identical to
that of BMV RNA3 except that the 3'-tenminal A will be omitted.
xample 4: Insertion of a bacterial chloramphenicol resistance gene in a BMV
RNA3 derivative a expression of a functional protein in barley_ _
cells
Plasmid pB3~P1 (Example 3) was cleaved with SalI and XbaI to delete most
of the coat protein gene except for seven nucleotides at the beginning of the
coat protein coding sequence including the AUG start codon, treated with the
Klenow fragment of DNA polymerase I to produce blunt ends and the resulting
larger DNA fragment isolated from a low melting point agarose gel. Plasmid
pBR325 (Bolivar, 1978) was digested with Taql, treated with Klenow polymerase
and the 780 bp fragment containing the chloramphenicol acyl transferase (CAT)
gene was isolated. The 780 bp fragment isolated in this manner contained the
entire CAT gene together with a short segment of pBR325 flanking the 5' end of
the CAT gene coding sequence. The larger pB3nP1 fragment and a three-fold
molar excess of the CAT fragment were ligated with T4 DNA 1igase and trans-
formed into E. coli JM101 cells. Plasmid DNA from selected ampicillin-
resistant transformants was screened by double digestion with EcoRI and PstI
and gel electrophoresis to confirm insertion of the CAT gene and to determine
its orientation with respect to BMV3 cDNA sequences. One plasmid, pB3CA42,
containing the CAT gene coding sequences in the same orientation as the BMV3
coding sequences was selected for further work along with a plasmid, pB3CA52,
with the CAT gene in the reverse orientation. Insertion of the CAT gene in
the positive orientation, as in pB3CA42, results in in-frame linkage of the
CAT coding sequences with the initiation codon of the BMN coat gene ~Fig.
3). Translation from the coat AUG would result in production of a fusion
protein bearing 12 additional amino acids before the start of the native CAT
gene product,
In a similar construction, diagrammed in Fig. 4, the same CAT fragment
was inserted at the SalI site of pB3~3 by SalI digestion followed by blunt
ending with Klenow polymerase and ligation with DNA ligase. Two clones
differing in the orientation of the CAT gene were isolated, pB3CA31 with the
CAT gene coding sequence oriented backwards from the direction of transcrip-
tion, and pB3CA21 with the CAT gene coding sequence oriented in the same
~38073
direction as that of transcription. Fig. 4 also shows the r~ucleotide sequence
in the region of the junction point between BMV-derived and bacterial-derived
sequences, for pB3CA21. As a control, SalI/XbaI deleted pB3 without an inser-
tion was constructed, designated pB3DCP, as shown in Fig. 5. The sequence in
the region of the subgenomic transcription start site and religation site is
also shown in Fig. 5. As a further control, the CAT coding sequence was
deleted from plasmid pB3CA42 (Fig. 3) by cleaving with SalI, filling out the
recessed 3' ends with Klenow DNA polymerase and deoxynucleotides, and religa-
ting the resultant blunt ends.
PstI-cut pB3CA42 DNA and EcoRI-cut pBlPM18 and pB2PM25 DNAs were tran-
scribed, LiCl-precipitated and used to inoculate protoplasts ~Example 1~.
After 22 hours incubation protoplasts were lysed by freezing and thawing and
were found to contain CAT activity as assayed by standard methods (Herrera-
Estrella et al. (1983) Nature 303, 209; Shaw, (1975) Methods Enzymol. 53,
737). Cell lysates were incubated with [14C] chloramphenicol and, following
the published procedure, silica gel thin layer plates separating reactants and
products were autoradiographed. The results are shown in Fig. 6. Lanes
marked Cm were loaded with [14C] chloramphenicol on1y. CAT activity in other
reactions is indicated by the appearance of acetylated chloramphenicol forms
marked lA-Cm (1-acetate) and 3A-Cm (3-acetate) in addition to the native form
marked Cm. The lanes marked CAT-mi and CAT show the products produced by
authentic bacterial CAT in the presence of extracts from mock-inoculated
protoplasts or buffer only. Panel A shows the products produced by extracts
obtained from protoplasts inocuiated with transcripts from pBlPM18 and
pB2PM25, together with pB3CA21 (lane designated CA 21), pB3CA31 (lane designa-
ted CA 31), pB3CA42 (lane designated CA 42), pB3CA52 (lane designated CA 52),
pB3CA61 (lane designated CA 61) or pB3PM1 (lane designated 3-1). In panel B
the products obtained from extracts of protoplasts inoculated with various
combinations of pBlPM18 (designated 1), pB2PM25 (designated 2), pB3PM1 (desig-
nated 3), and pB3CA42 (designated 3) are shown. In parallel tests, mock-
inoculated protoplasts and protoplasts inoculated with transcripts from EcoRI-
cut pBlPM18, EcoRI-cut pB2PM25 and either EcoRI-cut pB3PM1 or PstI-cut pB3CA52
showed no detectable CAT activity. The results shown in Fig. 6 demonstrate
phenotypic transformation of the cells and further demonstrate that an RNA-3'
containing an inserted nonviral coding segment, under appropriate conditions
of infection, can effect such transformation. Only the combination of 1 + 2
+ 3 provides expression of the CAT gene, showing that this expression is
dependent on viral RNA replication~
~l~88073
21
Example 5: Bal 31 Deletions in Plasmid pB3PM1
Plasmid pB3PM1 DNA was cleaved with ClaI, treated with T4 DNA polymerase
to produce blunt ends, and ligated to phosphorylated 12 bp synthetic BamHI
linkers (Maniatis et al., 1982). After phenol/chloroform extraction and
ethanol precipitation, the DNA was cleaved with 40 units BamHI per ~9 linker
for 16 hours at 37C. After electrophoresis on 1~ (w/v) low-melting point
agarose the major ethidium bromide-staining band of DNA was eluted (Sanger et
al., 1980) and recircularized by treatment with T4 DNA ligase at approximately
2 ng DNA/~l reaction, and transformed into competent E. coli JM101. RF DNA
from randomly selected ampicillin-resistant transformants was digested simul-
taneously with BamHI and EcoRI and screened by gel electrophoresis to confirm
the presence of the _ HI linker at the desired point. A single clone, desig-
nated pB3C49, was selected for further work.
12 ~9 of ClaI-cleaved pB3PM1 DNA was treated with 12 units of Bal 31 at
room temperature in a 180~1 reaction (Guo et al. (1983) Nucleic Acids Res. 11,
2237). 30~1 aliquots were removed 2, 4, 6, 8, 10 and 12 minutes after enzyme
addition. Nuclease digestion in each aliquot was terminated by addition of
25~1 of 40 mM EDTA and two successive phenol/chloroform extractions. The
aliquots were pooled and the DNA precipitated with ethanol. The DNA was
treated with the Klenow fragment of DNA polymerase I to generate blunt ends,
and 12 bp synthetic BamHI linkers were added (Maniatis et al, 1982). After
phenol/chloroform extraction and ethanol precipitation, the DNA was treated
with 50 units BamHI/~g linker and 2 units PstI/~g plasmid for 16 hours at
37C. Products were run on a low melting point agarose gel and the high MW
fraction containing the approximately 4.2 kb ClaI/PstI fragment of pB3PM1 and
its Bal 31-deleted, linker-ligated products was eluted and mixed with a molar
excess of the approximately 1.5 kb PstI-BamHI fragment of pB3C49. After
ligation, DNA was transformed into competent E. coli JM101 cells. RF DNA was
prepared from randomly-selected ampicillin-resistant transformants and was
screened by double digestion with BamHI and EcoRI followed by agarose gel
electrophoresis. Plasmids with deletions extending a variety of distances
from the initial ClaI site, within the 3a gene (Fig. 1), of pB3PM1 toward the
EcoRI site were selected using this data. Selected plasmids were cleaved with
EcoRI and transcribed (Example 1) and the transcripts used to infect barley
~ asts in the presence of transcripts from EcoRI-cut plasmids pBlPM18 and
807~
22
Using similar techniques a BamHI linker was inserted in the SacI site of
pB3P~1 and two further Bal 31 deletion libraries were constructed, one with
deletions extending 5' to the SacI site and one with deletions 3' to the SacI
site. Transcripts from selected EcoRI-cut plasmids were tested in the
presence of transcripts from EcoRI-cut pBlPM18 and pB3PM25 in the barley
protoplast system. Transcripts from pB3PMl derivatives with linker insertions
in either the ClaI and SacI sites, and from derivatives with deletions extend-
ing for up to several hundred bases from either site were found to replicate
under such conditions. Substantial deletions within the 3a gene and the coat
protein gene can therefore be made, at least several hundred bases from either
the ClaI site of the SacI site, without preventing replication of the deleted
RNA. Such deletions provide room for large insertions while still staying
within the size constraints for packaging replicated RNA3' into virus parti-
cles. The remaining portion of RNA, derived from RNA3, contains a c~s-acting
replication element of BMV RNA. Although the 3A gene and coat gene were not
required for RNA replication or for expression of the inserted CAT gene under
the conditions of infection used in the example, either or both of these genes
could, under other conditions, provide important secondary functions, for
example, by promoting systemic infection during transfer of whole plants.
Where deletion is not desired but the length of the modified RNA exceeds the
packaging constraints of the icosahedral BMV capsid, it may be possible to
provide for expression of the coat protein of a rod-shaped virus for encapsi-
dating the modified RNA.
Discussion and Conclusions
The foregoing examples demonstrate that substantial modifications, both
deletions and insertions, can be made in an RNA component of a multipartite
RNA virus without preventing replication of viral RNA under appropriate condi-
tions of infection. Genetic material inserted within a region of an RNA virus
that is nonessential for RNA replication is translatable. In the case of 8MV,
substantial portions of RNA3 can be deleted without loss of the ability to
replicate. Therefore any gene inserted within a nonessential region of an RNA
component of an RNA multipartite virus can be translated in the transfonmed
cel1, provided the gene has appropriate ribosome binding and translation
initiation signals at its 5' end. These signals can be provided by the virus
or by the insert and the means for making translatable constructions is within
the scope of capability oF those ordinarily skilled in the art.
~l~8~3073
23
While the foregoing principles were illustrated in the case of BMV RNA3,
it is apparent that any component of any RNA virus is a candidate for modifi-
cations of the type illustrated. For example, an exogenous RNA segment could
be inserted at any site of BMN RNA 1 or 2 which does not result in loss of
ability to replicate. Similarly, the RNA components of other RNA viruses can
be similarly manipulated, provided the insertions and/or deletions employed do
not prevent replication of viral RNA. The two operating principles which
permit the modification of a viral RNA component to make it a vector for
carrying translatable genetic material into a host cell are: (1) cloning a
cDNA of the RNA component into a transcription vector capable of transcribing
replicatable RNA from viral cDNA, and (2) the identification of a region in
one of the viral components that is nonessential for replication, into which a
structural gene can be inserted. ~he modified RNA will therefore contain, at
a minimum, a cis-acting replication element derived from an RNA virus and an
inserted exogenous RNA segment. Further modifications and improvements
following and embodying the teachings and disclosures herein are deemed to be
within the scope of the invention, as set forth in the appended claims.
